An Examination of Chemical Tools for Hydrogen Selenide Donation and Detection

Hydrogen selenide (H2Se) is an emerging biomolecule of interest with similar properties to that of other gaseous signaling molecules (i.e., gasotransmitters that include nitric oxide, carbon monoxide, and hydrogen sulfide). H2Se is enzymatically generated in humans where it serves as a key metabolic intermediate in the production of selenoproteins and other selenium-containing biomolecules. However, beyond its participation in biosynthetic pathways, its involvement in cellular signaling or other biological mechanisms remains unclear. To uncover its true biological significance, H2Se-specific chemical tools capable of functioning under physiological conditions are required but lacking in comparison to those that exist for other gasotransmitters. Recently, researchers have begun to fill this unmet need by developing new H2Se-releasing compounds, along with pioneering methods for selenide detection and quantification. In combination, the chemical tools highlighted in this review have the potential to spark groundbreaking explorations into the chemical biology of H2Se, which may lead to its branding as the fourth official gasotransmitter.


Introduction
The discovery of elemental selenium can be traced back to a Swedish sulfuric acid plant in the early 19th Century where chemist Jöns Jakob Berzelius first observed a reddishbrown sediment in the acid being produced there [1].Berzelius initially mistook this new substance for tellurium due to its odor and appearance.Famed for his advancement of modern chemical notation, the principle of stoichiometry, and the determination of atomic weights of most known elements at the time, Berzelius chemically compared the red-brown byproduct with a known sample of tellurium and determined that the two were, in fact, different elements with the new substance having the properties of a metal combined with that of sulfur.In the words of Berzelius, he had discovered "a new kind of sulfur".
Today, selenium is regarded as an essential micronutrient that is acquired through dietary means with an optimal daily dose of 55 µg for adults [2].There are at least 25 selenoproteins [3][4][5], many of which play a central role in cellular redox homeostasis, including glutathione peroxidases (GPx) and thioredoxin reductases (TrxR), and require dietary selenium for their production.Low selenium levels in humans are associated with a myriad of illnesses [6][7][8][9].Keshan Disease [10,11], a potentially fatal form of cardiomyopathy, is primarily observed in selenium-deficient regions in China.The same is true for Kashin-Beck Disease [12,13], a chronic joint disease predominantly found in parts of the world where selenium is scarce.Weakened immune function [14,15], cardiovascular diseases [16,17], and certain cancers [18][19][20][21] are also strongly associated with selenium deficiency.These correlative studies suggest that the role of selenium in human health and biology could extend beyond its incorporation into selenium-containing proteins and point towards its possible involvement in other cellular processes.
H 2 S is primarily produced by three principal enzymes-cystathionine β-synthase (CBS) [31], cystathionine γ-lyase (CSE) [32], and 3-mercaptopyruvate sulfur transferase (3-MST) [33]-providing exquisite control over its production.As such, endogenous H 2 S is known to be involved in numerous signaling processes throughout the body, including the brain and central nervous system [34,35], and within specific cellular compartments (i.e., mitochondria) [36,37].Much of what is known about H 2 S pharmacology stems from the advent of donor compounds, synthetic small molecules designed to slowly liberate H 2 S in a controlled fashion that mimics its natural biosynthesis, and the use of said compounds in various cellular and animal models of disease [38][39][40][41].
Alongside nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide is the most recent, widely recognized member of the gasotransmitter family [42][43][44].Its inclusion was suggested in the early 2000s and was based on five key observations [45].Namely, H 2 S is (i) a small molecule gas that (ii) freely permeates cellular membranes.It is (iii) endogenously and enzymatically generated, with (iv) well-defined biological functions that stem from its (v) action at specific cellular targets.
It is interesting to note that H 2 Se (Predominantly HSe − at physiological pH) already checks several of these boxes.It exists as a lipophilic gas in its diprotic form and is expressed enzymatically in mammals where it serves as a key intermediate in the production of selenium-containing biomolecules (Figure 1) [46].H 2 Se was shown to act on at least some protein targets, including inhibition of cytochrome c oxidase, which modulates aerobic respiration [46,47].Moreover, it is the reduced selenide form of selenium, and not the often administrated selenite (or other oxidized forms), that is believed to be responsible for the observed biological activity of selenium, including its anticancer effects and noted protection against myocardial ischemia reperfusion injury [48][49][50].
H2S is primarily produced by three principal enzymes-cystathionine β-synthase (CBS) [31], cystathionine γ-lyase (CSE) [32], and 3-mercaptopyruvate sulfur transferase (3-MST) [33]-providing exquisite control over its production.As such, endogenous H2S is known to be involved in numerous signaling processes throughout the body, including the brain and central nervous system [34,35], and within specific cellular compartments (i.e., mitochondria) [36,37].Much of what is known about H2S pharmacology stems from the advent of donor compounds, synthetic small molecules designed to slowly liberate H2S in a controlled fashion that mimics its natural biosynthesis, and the use of said compounds in various cellular and animal models of disease [38][39][40][41].
Alongside nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide is the most recent, widely recognized member of the gasotransmi er family [42][43][44].Its inclusion was suggested in the early 2000s and was based on five key observations [45].Namely, H2S is (i) a small molecule gas that (ii) freely permeates cellular membranes.It is (iii) endogenously and enzymatically generated, with (iv) well-defined biological functions that stem from its (v) action at specific cellular targets.
It is interesting to note that H2Se (Predominantly HSe − at physiological pH) already checks several of these boxes.It exists as a lipophilic gas in its diprotic form and is expressed enzymatically in mammals where it serves as a key intermediate in the production of selenium-containing biomolecules (Figure 1) [46].H2Se was shown to act on at least some protein targets, including inhibition of cytochrome c oxidase, which modulates aerobic respiration [46,47].Moreover, it is the reduced selenide form of selenium, and not the often administrated selenite (or other oxidized forms), that is believed to be responsible for the observed biological activity of selenium, including its anticancer effects and noted protection against myocardial ischemia reperfusion injury [48][49][50].Still, compared to H 2 S, very little is known about the (patho)physiological effects of H 2 Se.This is due, at least in part, to a lack of refined donor compounds that can increase the bioavailability of selenide and be used to effectively modulate cellular concentrations.We and others have begun to fill this unmet need by developing new H 2 Se-releasing compounds, which we will highlight below, along with current methods for selenide detection and quantification.In combination, the chemical tools outlined in this review have the potential to serve as invaluable exploratory tools for uncovering H 2 Se biology and its potential in medicine.

Selenotrisulfides
A key intermediate in the biosynthesis of H 2 Se is believed to be glutathione selenotrisulfide (GSSeSG), which forms in vitro from the reduction of selenite with four equivalents of glutathione (GSH) [51][52][53].While the isolation of GSSeSG is difficult due to its chemical instability, the reduction of selenite is not specific to GSH, meaning the preparation and evaluation of alternative selenotrisulfides (RSSeSR) as potential H 2 Se-donating motifs could be a viable option (Figure 2A).Still, compared to H2S, very li le is known about the (patho)physiological effects of H2Se.This is due, at least in part, to a lack of refined donor compounds that can increase the bioavailability of selenide and be used to effectively modulate cellular concentrations.We and others have begun to fill this unmet need by developing new H2Se-releasing compounds, which we will highlight below, along with current methods for selenide detection and quantification.In combination, the chemical tools outlined in this review have the potential to serve as invaluable exploratory tools for uncovering H2Se biology and its potential in medicine.

Selenotrisulfides
A key intermediate in the biosynthesis of H2Se is believed to be glutathione selenotrisulfide (GSSeSG), which forms in vitro from the reduction of selenite with four equivalents of glutathione (GSH) [51][52][53].While the isolation of GSSeSG is difficult due to its chemical instability, the reduction of selenite is not specific to GSH, meaning the preparation and evaluation of alternative selenotrisulfides (RSSeSR) as potential H2Se-donating motifs could be a viable option (Figure 2A).In a pioneering study by Nakayama and co-workers, the more robust penicillamine selenotrisulfide (PenSSeSPen) was prepared, which is isolable due to increased steric bulk near the S-Se-S motif, and its bioavailability was compared with that of selenite, an established dietary source of selenium, in Se-deficient mice [54].Following oral administration, the selenium content in selected organs was quantified fluorometrically using 2,3-diaminonaphthalene.Similar to selenite-treated mice, the administration of PenSSeSPen led to a significant increase in selenium in the heart, liver and blood.Additionally, PenSSeSPen-fed mice exhibited similar GPx activity to that of selenite-fed mice, indicating that the selenium content from PenSSeSPen was available for selenoprotein production.
In a later study by Nakayama, it was proposed that human serum albumin (HSA), the most abundant plasma protein, serves as a selenium carrier via a selenotrisulfide linkage that enables it to distribute selenide to peripheral tissues and organs throughout the body [55].The authors observed that treatment of red blood cells (RBCs) with selenite led to selenium efflux that was dependent on HSA concentration.Moreover, pretreatment of HSA with iodoacetamide, a thiol-blocking agent, appeared to inhibit selenium transfer from RBCs to HSA, confirming that the thiol functional group on HSA played a key role in the transfer event.When selenium-bound HSA was treated with penicillamine (Pen), selenotrisulfide PenSSeSPen was produced.Additionally, the same PenSSeSPen product was formed when the selenium efflux experiment was conducted in the presence of Pen rather than HSA.These observations led to the conclusion that selenium is likely to be exported from RBCs as a selenotrisulfide (RSSeSR), which forms from the reaction between selenite and an RBC thiol.The ensuing selenotrisulfide then binds to HSA via a thiol exchange reaction, enabling the transport of selenium throughout the body (Figure 2B).In a pioneering study by Nakayama and co-workers, the more robust penicillamine selenotrisulfide (PenSSeSPen) was prepared, which is isolable due to increased steric bulk near the S-Se-S motif, and its bioavailability was compared with that of selenite, an established dietary source of selenium, in Se-deficient mice [54].Following oral administration, the selenium content in selected organs was quantified fluorometrically using 2,3-diaminonaphthalene.Similar to selenite-treated mice, the administration of PenSS-eSPen led to a significant increase in selenium in the heart, liver and blood.Additionally, PenSSeSPen-fed mice exhibited similar GPx activity to that of selenite-fed mice, indicating that the selenium content from PenSSeSPen was available for selenoprotein production.
In a later study by Nakayama, it was proposed that human serum albumin (HSA), the most abundant plasma protein, serves as a selenium carrier via a selenotrisulfide linkage that enables it to distribute selenide to peripheral tissues and organs throughout the body [55].The authors observed that treatment of red blood cells (RBCs) with selenite led to selenium efflux that was dependent on HSA concentration.Moreover, pretreatment of HSA with iodoacetamide, a thiol-blocking agent, appeared to inhibit selenium transfer from RBCs to HSA, confirming that the thiol functional group on HSA played a key role in the transfer event.When selenium-bound HSA was treated with penicillamine (Pen), selenotrisulfide PenSSeSPen was produced.Additionally, the same PenSSeSPen product was formed when the selenium efflux experiment was conducted in the presence of Pen rather than HSA.These observations led to the conclusion that selenium is likely to be exported from RBCs as a selenotrisulfide (RSSeSR), which forms from the reaction between selenite and an RBC thiol.The ensuing selenotrisulfide then binds to HSA via a thiol exchange reaction, enabling the transport of selenium throughout the body (Figure 2B).
In a very recent study by Wang, Xu, Xie, and co-workers, it was reported that a stable selenotrisulfide (AcidSSeSAcid) could be formed by treating 2-mercaptoacetic acid with selenium dioxide [56].However, when the same reaction was run with 2-mercaptoethanol, the resultant selenotrisulfide (HydSSeSHyd) behaved more like GSSeSG and proved difficult to characterize.Thus, only the reactivity of the more robust AcidSSeSAcid was assessed further in the presence of glutathione.Using ESI-MS, the byproducts of this reaction were found to be GSSG, AcidSH, AcidSSeSG, AcidSSG, and AcidSSeH, providing some indirect evidence of glutathione-promoted H 2 Se release.At the outset of this study, the authors hypothesized that H 2 Se might function as an "H 2 S disguiser" and thereby assist in overcoming H 2 S-indcued antibiotic resistance.This theory was tested with AcidSSeSAcid in an H 2 S-induced antibiotic-resistant MRSA model (MRSA S+ ).While the antibiotic gentamicin alone proved ineffective against MRSA S+ it, in combination with AcidSSeSAcid, displayed impressive bactericidal activity.The authors attributed this reduction in antibiotic resistance to the release of H 2 Se, which increases bacterial membrane permeability and reactivates bacterial respiratory flux.
Although the direct liberation of H 2 Se was not confirmed in any of these studies with trapping experiments, they do underscore the biological relevance of the RSSeSR motif and its likely ability to function as an endogenous selenide delivery agent.The general instability of this framework, however, may limit its overall utility as an exogenous source of H 2 Se, as the ability to generate a large library of selenotrisulfides with tunable rates of release could prove difficult.Thus, the search continues for alternative frameworks with the potential to supply selenide in a controlled and sustained fashion.

Selenide Salts
Logically, many H 2 Se donors were inspired by previously reported H 2 S-releasing compounds.To this end, selenide salts, which serve as a convenient H 2 Se equivalent in buffer, were examined for convenience [47,50,57], drawing parallels to sulfide salts being used in initial studies aimed at exploring H 2 S chemical biology [58].
Using sodium hydroselenide (NaHSe), the Dyson group was among the first to explore in detail the pharmacology and therapeutic utility of H 2 Se [47].Given its instability and absence of a reliable commercial source, the group elected to generate NaHSe in-house by reducing elemental selenium with an aqueous solution of sodium borohydride [59].Once in hand, they evaluated the metabolic effects of NaHSe ex vivo using dissected rat soleus muscle and homogenized liver tissue.In these models, NaHSe was shown to inhibit oxygen consumption in a concentration-dependent manner, albeit to a lesser extent than sodium hydrosulfide (NaHS) and potassium cyanide (KCN), which were used as positive controls.The authors also investigated the mechanism of inhibition of O 2 consumption and found its inhibition of cytochrome C oxidase to be a likely candidate, similar to NaHS.
The influence of NaHSe on selenoprotein expression in HepG2 (human hepatocyte) cells was also inspected [47].Using a Western blot analysis, a significant increase in the production of glutathione peroxidase-1 (GPx-1) was observed upon treatment with NaHSe.Interestingly, the addition of selenite (SeO 3 2− ), a common dietary source of selenium, had the opposite effect with a notable reduction in GPx-1 expression compared to nontreated controls.Moreover, the Dyson group also demonstrated that the addition of DL-propargylglycine (PAG), an established CSE inhibitor, led to significant reductions in both GPx-1 and thioredoxin reductase-1 (TrxR), presumably due to its inhibition of the H 2 Se producing enzyme selenocysteine lyase (SCLY, Figure 1).However, the expression of both proteins was restored by the addition of exogenous NaHSe, suggesting that endogenous selenoprotein expression can be regulated by H 2 Se administration.
The therapeutic value of H 2 Se supplementation was also examined in HepG2 cells exposed to hydrogen peroxide (H 2 O 2 ) insult [47].Indeed, incubation with NaHSe (0.3 and 1 µM) for 1 h conferred cellular protection in a dose-dependent manner.The authors noted that multiple mechanisms could be in play to account for the improved ROS management by NaHSe-treated cells, including its direct ROS scavenging, its functioning as a metabolic modulator, and/or its serving as the catalytic component of antioxidant selenoproteins.
Finally, the in vivo pharmacological effects of NaHSe were also investigated in this study [47].Anesthetized rats were given an escalated dose of 0.01 mg/kg to 10 mg/kg of selenide.Both blood pressure and heart rate decreased notably at the highest dose level but, overall, cardiac output remained relatively unaffected.The authors also noted significant hyperlactatemia (inhibition of oxidative phosphorylation) at the highest dose of NaHSe.
To date, this remains one of the most comprehensive studies, detailing the chemical biology and pharmacological effects of H 2 Se [47].Still, a severe limitation, even noted by the authors, was the use of NaHSe as an H 2 Se source.As cited above, the use of selenide salts is analogous to early work on H 2 S which employed sulfide salts as a convenient method for H 2 S delivery.Their use, however, creates a bolus effect that poorly mimics the endogenic production of H 2 S. The same is likely true for the use of selenide salts as a research tool for examining the pharmacology and medicinal value of H 2 Se.Thus, refined selenium-containing compounds with suitable pharmacokinetics and exquisite control over their selenide release are highly desirable and early attempts to access such compounds are summarized below.

Selenoanhydrides
Selenoanhydrides were among the first small molecules assessed for their ability to deliver selenide in a controlled fashion and in response to biologically pertinent molecules [60].Domínguez-Álvarez and co-workers reported on the impressive anticancer effects of selenoanhydride R-Se (Figure 3A) in earlier studies and suspected it might be due to its release of H 2 Se [61][62][63].To examine this, they monitored the fragmentation pattern of R-Se in a 50% methanol/water solution.Under electrospray ionization conditions (ESI), initial attack by methanol led to the observed fragmentation products with the loss of H 2 Se (Figure 3B).Moreover, the addition of Na 2 S appeared to amplify the decomposition of the donor with selenium-containing fragments being observed but with a significantly lower abundance.While experimental conditions were not biologically relevant, this simple ESI-MS analysis did provide some insight into the propensity of selenoanhydrides to expel selenide upon nucleophilic exposure.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 21 by NaHSe-treated cells, including its direct ROS scavenging, its functioning as a metabolic modulator, and/or its serving as the catalytic component of antioxidant selenoproteins.Finally, the in vivo pharmacological effects of NaHSe were also investigated in this study [47].Anesthetized rats were given an escalated dose of 0.01 mg/kg to 10 mg/kg of selenide.Both blood pressure and heart rate decreased notably at the highest dose level but, overall, cardiac output remained relatively unaffected.The authors also noted significant hyperlactatemia (inhibition of oxidative phosphorylation) at the highest dose of NaHSe.
To date, this remains one of the most comprehensive studies, detailing the chemical biology and pharmacological effects of H2Se [47].Still, a severe limitation, even noted by the authors, was the use of NaHSe as an H2Se source.As cited above, the use of selenide salts is analogous to early work on H2S which employed sulfide salts as a convenient method for H2S delivery.Their use, however, creates a bolus effect that poorly mimics the endogenic production of H2S.The same is likely true for the use of selenide salts as a research tool for examining the pharmacology and medicinal value of H2Se.Thus, refined selenium-containing compounds with suitable pharmacokinetics and exquisite control over their selenide release are highly desirable and early a empts to access such compounds are summarized below.

Selenoanhydrides
Selenoanhydrides were among the first small molecules assessed for their ability to deliver selenide in a controlled fashion and in response to biologically pertinent molecules [60].Domínguez-Álvarez and co-workers reported on the impressive anticancer effects of selenoanhydride R-Se (Figure 3A) in earlier studies and suspected it might be due to its release of H2Se [61][62][63].To examine this, they monitored the fragmentation pa ern of R-Se in a 50% methanol/water solution.Under electrospray ionization conditions (ESI), initial a ack by methanol led to the observed fragmentation products with the loss of H2Se (Figure 3B).Moreover, the addition of Na2S appeared to amplify the decomposition of the donor with selenium-containing fragments being observed but with a significantly lower abundance.While experimental conditions were not biologically relevant, this simple ESI-MS analysis did provide some insight into the propensity of selenoanhydrides to expel selenide upon nucleophilic exposure.The reducing capacity of R-Se and related chalcogens (R-S and R-O, Figure 3A) was assessed using cPTIO, a nitric oxide radical scavenger [60].The authors noted that the addition of H 2 S potentiated the radical scavenging ability of R-Se and R-S (albeit to a lesser extent) but not R-O.The same trend was observed with the antioxidant glutathione (GSH).While the addition of GSH alone did not effectively quench cPTIO, it in combination with R-Se was found to significantly reduce cPTIO radicals.Similar results were obtained with superoxide (O 2 •− ).Using BMPO as an EPR spin trap reagent, the authors found that H 2 S/R-Se and H 2 S/R-S (but not H 2 S/R-O) could effectively scavenge BMPO-OOH/OH adducts and, perhaps, O 2 •− directly.In total, these observations indicate that the reducing potency of thiols (i.e., GSH and H 2 S) are significantly boosted upon their interaction with R-Se (and to a lesser extent R-S), which is likely to liberate reactive selenium (or sulfur) species, including hydrogen selenide.Although these studies never provided any direct evidence of H 2 Se release from R-Se, the radical scavenging activity of this purported donor provides a strong indication.

P=Se Motifs
With inspiration from GYY4137, an early and extensively studied H 2 S donor that gradually decomposes to release H 2 S via P=S bond cleavage [64], Pluth and co-workers developed an analogous H 2 Se donor, TDN1042, that delivered selenide (H 2 S/H 2 Se − ) via a similar hydrolytic pathway (Figure 4) [65].Synthetically, TD1042 was accessed upon treatment of Woollins' reagent with excess morpholine, akin to the preparation of GYY4137.The reducing capacity of R-Se and related chalcogens (R-S and R-O, Figure 3A) was assessed using cPTIO, a nitric oxide radical scavenger [60].The authors noted that the addition of H2S potentiated the radical scavenging ability of R-Se and R-S (albeit to a lesser extent) but not R-O.The same trend was observed with the antioxidant glutathione (GSH).While the addition of GSH alone did not effectively quench cPTIO, it in combination with R-Se was found to significantly reduce cPTIO radicals.Similar results were obtained with superoxide (O2 •− ).Using BMPO as an EPR spin trap reagent, the authors found that H2S/R-Se and H2S/R-S (but not H2S/R-O) could effectively scavenge BMPO-OOH/OH adducts and, perhaps, O2 •− directly.
In total, these observations indicate that the reducing potency of thiols (i.e., GSH and H2S) are significantly boosted upon their interaction with R-Se (and to a lesser extent R-S), which is likely to liberate reactive selenium (or sulfur) species, including hydrogen selenide.Although these studies never provided any direct evidence of H2Se release from R-Se, the radical scavenging activity of this purported donor provides a strong indication.

P=Se Motifs
With inspiration from GYY4137, an early and extensively studied H2S donor that gradually decomposes to release H2S via P=S bond cleavage [64], Pluth and co-workers developed an analogous H2Se donor, TDN1042, that delivered selenide (H2S/H2Se − ) via a similar hydrolytic pathway (Figure 4) [65].Synthetically, TD1042 was accessed upon treatment of Woollins' reagent with excess morpholine, akin to the preparation of GYY4137.Once prepared and structurally characterized by NMR and single crystal X-ray diffraction, H2Se release from TDN1042 in wet DMSO was evaluated using 31 P NMR spectroscopy.These studies revealed the clean conversation of TD1042 to phenylphosphonic acid, as expected (Figure 4).This clean transformation to phenylphosphonic acid was also observed in citrate buffer (pH 3.0 to 6.0), with higher rates of release occurring at more acidic pH values, which is consistent with a hydrolysis-based mechanism.
The authors also confirmed the direct liberation of H2Se from TD1042, a key experiment that was omi ed from previous reports of H2Se donating motifs.To accomplish this, an aqueous solution of TD1042 was acidified with HCl and sparged with N2 to assist in volatilizing any released H2Se into the vial headspace where it was then bubbled through a separate trapping solution of dinitrofluorobenzene (DNFB).Using HPLC, Pluth and coworkers observed the formation of both di(2,4-dinitrophenyl) selenide ((DNP)2Se) and the related diselenide ((DNP)2Se2) in the trapping solution.
In a later study, the Pluth group sought to augment the rate of H2Se donation from this platform through the introduction of cyclic-PSe donors.[66] To accomplish this, Woollins' reagent was treated with various ortho-substituted phenols to generate a small library of donors with a single P=Se motif (Cat-PSe, 2AP-PSe, and NMe2AP-PSe, Figure 5A).Once prepared and structurally characterized by NMR and single crystal X-ray diffraction, H 2 Se release from TDN1042 in wet DMSO was evaluated using 31 P NMR spectroscopy.These studies revealed the clean conversation of TD1042 to phenylphosphonic acid, as expected (Figure 4).This clean transformation to phenylphosphonic acid was also observed in citrate buffer (pH 3.0 to 6.0), with higher rates of release occurring at more acidic pH values, which is consistent with a hydrolysis-based mechanism.
The authors also confirmed the direct liberation of H 2 Se from TD1042, a key experiment that was omitted from previous reports of H 2 Se donating motifs.To accomplish this, an aqueous solution of TD1042 was acidified with HCl and sparged with N 2 to assist in volatilizing any released H 2 Se into the vial headspace where it was then bubbled through a separate trapping solution of dinitrofluorobenzene (DNFB).Using HPLC, Pluth and co-workers observed the formation of both di(2,4-dinitrophenyl) selenide ((DNP) 2 Se) and the related diselenide ((DNP) 2 Se 2 ) in the trapping solution.
In a later study, the Pluth group sought to augment the rate of H 2 Se donation from this platform through the introduction of cyclic-PSe donors [66].To accomplish this, Woollins' reagent was treated with various ortho-substituted phenols to generate a small library of donors with a single P=Se motif (Cat-PSe, 2AP-PSe, and NMe2AP-PSe, Figure 5A).By introducing a second electronegative heteroatom (i.e., oxygen or nitrogen), it was suspected that the rate of hydrolysis would increase due to the enhanced electrophilic character of the phosphorous center.This was confirmed by 31 P NMR, which was used to monitor donor hydrolysis in PIPES buffer (pH 7.4).Like TD1042, cyclic-PSe compounds were found to release selenide while cleanly forming phenylphosphonic acid as a byproduct (Figure 5B).However, unlike TD1042, these donors were shown to operate at neutral pH due to their enhanced hydrolytic lability.The rates of hydrolysis among cyclic-PSe donors varied significantly with Cat-PSe displaying the slowest rate of hydrolysis and 2AP-PSe hydrolyzing the quickest.This result was somewhat unexpected and suggests that factors other than electronic effects influence the rates of hydrolysis.
Cell permeability studies were also conducted using time-of-flight secondary ion mass spectrometry (TOF-SIMS).A dose-dependent increase in intracellular selenium levels was observed in HeLa cells with increasing concentrations of 2AP-PSe, the most efficient H2Se donor identified in this study.
The in cellulo antioxidant activity of 2AP-PSe was also assessed.When live HeLa cells were treated with exogenous H2O2 (500 µM) and peroxide sensor DCFH-DA, a significant fluorescence response was observed due to the formation of 2′,7′-dichlorofluorescein (DCF) [67].However, when cells were first pre-treated with 2AP-PSe (5-25 µM) a notable decrease in ROS-generated fluorescence was observed.Due to the instability of H2Se, the authors stress that the observed effects are unlikely due to the buildup of H2Se, but rather an increase in selenocompounds (likely antioxidant proteins) that effectively scavenge ROS.

Selenocarbonyls
In a comprehensive study by Yi and co-workers, both selenocyclopropenones and arylselenoamides were found to provide highly tunable rates of H2Se delivery in the presence of supraphysiological concentrations of cysteine [68].
Initially, the authors generated a selenium analogue of Michler's ketone but found that it quickly hydrolyzed in a buffer, producing a red residue (Se 0 ), presumably due to its rapid discharge of selenide (Figure 6A).Searching for a selenocarbonyl with a more tractable H2Se-releasing profile, selenocyclopropenones were then investigated.A small library was generated by treating the corresponding ketone with Woollins' reagent (Figure 6B).Compound 1 was chosen for initial studies, evaluating its reactivity and selectivity as a selenide donor.HPLC analysis confirmed that 1 was stable in a 50% PBS/CH3CN (pH 7.4) mixture.The introduction of cysteine (2-10 mM), however, led to the expulsion of selenide and the subsequent formation of a red solid (Se 0 ).This was further corroborated by an H2Se-selective gas detector.Donors 2 and 3, with electron-withdrawing substituents at the para position, were found to liberate even more H2Se than 1 in the presence By introducing a second electronegative heteroatom (i.e., oxygen or nitrogen), it was suspected that the rate of hydrolysis would increase due to the enhanced electrophilic character of the phosphorous center.This was confirmed by 31 P NMR, which was used to monitor donor hydrolysis in PIPES buffer (pH 7.4).Like TD1042, cyclic-PSe compounds were found to release selenide while cleanly forming phenylphosphonic acid as a byproduct (Figure 5B).However, unlike TD1042, these donors were shown to operate at neutral pH due to their enhanced hydrolytic lability.The rates of hydrolysis among cyclic-PSe donors varied significantly with Cat-PSe displaying the slowest rate of hydrolysis and 2AP-PSe hydrolyzing the quickest.This result was somewhat unexpected and suggests that factors other than electronic effects influence the rates of hydrolysis.
Cell permeability studies were also conducted using time-of-flight secondary ion mass spectrometry (TOF-SIMS).A dose-dependent increase in intracellular selenium levels was observed in HeLa cells with increasing concentrations of 2AP-PSe, the most efficient H 2 Se donor identified in this study.
The in cellulo antioxidant activity of 2AP-PSe was also assessed.When live HeLa cells were treated with exogenous H 2 O 2 (500 µM) and peroxide sensor DCFH-DA, a significant fluorescence response was observed due to the formation of 2 ′ ,7 ′ -dichlorofluorescein (DCF) [67].However, when cells were first pre-treated with 2AP-PSe (5-25 µM) a notable decrease in ROS-generated fluorescence was observed.Due to the instability of H 2 Se, the authors stress that the observed effects are unlikely due to the buildup of H 2 Se, but rather an increase in selenocompounds (likely antioxidant proteins) that effectively scavenge ROS.

Selenocarbonyls
In a comprehensive study by Yi and co-workers, both selenocyclopropenones and arylselenoamides were found to provide highly tunable rates of H 2 Se delivery in the presence of supraphysiological concentrations of cysteine [68].
Initially, the authors generated a selenium analogue of Michler's ketone but found that it quickly hydrolyzed in a buffer, producing a red residue (Se 0 ), presumably due to its rapid discharge of selenide (Figure 6A).Searching for a selenocarbonyl with a more tractable H 2 Se-releasing profile, selenocyclopropenones were then investigated.A small library was generated by treating the corresponding ketone with Woollins' reagent (Figure 6B).Compound 1 was chosen for initial studies, evaluating its reactivity and selectivity as a selenide donor.HPLC analysis confirmed that 1 was stable in a 50% PBS/CH 3 CN (pH 7.4) mixture.The introduction of cysteine (2-10 mM), however, led to the expulsion of selenide and the subsequent formation of a red solid (Se 0 ).This was further corroborated by an H 2 Se-selective gas detector.Donors 2 and 3, with electron-withdrawing substituents at the para position, were found to liberate even more H 2 Se than 1 in the presence of cysteine, implying that the rates of donation from this platform can be easily tuned via simple structural modifications that alter the electrophilicity of the selenocarbonyl.The reaction between 1 and cysteine was further scrutinized by high-resolution mass spectrometry (HRMS) and identifiable byproducts were uncovered.Based on these observations, a mechanism for cys/thiol-triggered H 2 Se release from selenocyclopropenones was put forward by the authors (Figure 6B).
Molecules 2024, 29, x FOR PEER REVIEW 8 of 21 of cysteine, implying that the rates of donation from this platform can be easily tuned via simple structural modifications that alter the electrophilicity of the selenocarbonyl.The reaction between 1 and cysteine was further scrutinized by high-resolution mass spectrometry (HRMS) and identifiable byproducts were uncovered.Based on these observations, a mechanism for cys/thiol-triggered H2Se release from selenocyclopropenones was put forward by the authors (Figure 6B).Aryl thioamides were previously shown to function as an advantageous platform for the controlled delivery of H 2 S under biologically relevant conditions [69][70][71].Yi and co-workers suspected that selenoanalogues would provide an avenue for H 2 Se donation under similar conditions (Figure 6C) [68].To test their hypothesis, a small library of selenoamides was generated by treating benzamide derivatives with Woollins' reagent or by subjecting 4-hydroxybenzonitrile to a mixture of selenium powder and NaBH 4 .Selenobenzamide (4) was shown to be stable in PBS (pH 7.4), but the addition of cysteine led to the formation of red Se 0 .This was further substantiated with an H 2 Se gas detector.As expected, the rate of H 2 Se release from 4 was found to be significantly slower than 1.However, it was noted that the selenoamide platform could be structurally modified to alter reaction kinetics.For example, the introduction of an electron-donating hydroxyl group at the para position on the phenyl ring (5, Figure 6C) appeared to amplify the speed of selenide release.However, when the hydroxyl group was converted to a methyl ester (6, Figure 6C), its donating efficiency diminished somewhat, even relative to 4. On the other hand, amide N-alkylation (7, Figure 6C) was shown to suppress the rate of selenide delivery even further.HPLC analysis and DFT calculations were used to establish a mechanism for cysteinetriggered H 2 Se donation (Figure 6C).This proposed pathway aligns with the observation that treatment of arylselenoamides with other thiols that lack a nucleophilic amine (i.e., N-acetylcysteine, glutathione, and β-mercaptoethanol) fail to generate H 2 Se.

Selenocarbamates
Caged thiocarbamates have offered a reliable avenue for H 2 S delivery (Figure 7A).They also provide an opportunity to selectivity tune the release of H 2 S to a specific biological trigger (i.e., ROS [72][73][74][75][76], light [77,78], pH [79,80], and enzymes [81,82]).However, the production of H 2 S from this donor class is multi-layered, with the triggering event causing the donor to undergo a self-immolating process that first generates carbonyl sulfide (COS) prior to its quick conversation to H 2 S by the ubiquitous enzyme carbonic anhydrase (Figure 7A) [83].
Molecules 2024, 29, x FOR PEER REVIEW 9 of 21 Aryl thioamides were previously shown to function as an advantageous platform for the controlled delivery of H2S under biologically relevant conditions [69][70][71].Yi and coworkers suspected that selenoanalogues would provide an avenue for H2Se donation under similar conditions (Figure 6C) [68].To test their hypothesis, a small library of selenoamides was generated by treating benzamide derivatives with Woollins' reagent or by subjecting 4-hydroxybenzonitrile to a mixture of selenium powder and NaBH4.Selenobenzamide (4) was shown to be stable in PBS (pH 7.4), but the addition of cysteine led to the formation of red Se 0 .This was further substantiated with an H2Se gas detector.As expected, the rate of H2Se release from 4 was found to be significantly slower than 1.However, it was noted that the selenoamide platform could be structurally modified to alter reaction kinetics.For example, the introduction of an electron-donating hydroxyl group at the para position on the phenyl ring (5, Figure 6C) appeared to amplify the speed of selenide release.However, when the hydroxyl group was converted to a methyl ester (6, Figure 6C), its donating efficiency diminished somewhat, even relative to 4. On the other hand, amide N-alkylation (7, Figure 6C) was shown to suppress the rate of selenide delivery even further.HPLC analysis and DFT calculations were used to establish a mechanism for cysteine-triggered H2Se donation (Figure 6C).This proposed pathway aligns with the observation that treatment of arylselenoamides with other thiols that lack a nucleophilic amine (i.e., N-acetylcysteine, glutathione, and β-mercaptoethanol) fail to generate H2Se.

Selenocarbamates
Caged thiocarbamates have offered a reliable avenue for H2S delivery (Figure 7A).They also provide an opportunity to selectivity tune the release of H2S to a specific biological trigger (i.e., ROS [72][73][74][75][76], light [77,78], pH [79,80], and enzymes [81,82]).However, the production of H2S from this donor class is multi-layered, with the triggering event causing the donor to undergo a self-immolating process that first generates carbonyl sulfide (COS) prior to its quick conversation to H2S by the ubiquitous enzyme carbonic anhydrase (Figure 7A) [83].Continuing with the common theme of prior H 2 S-releasing motifs laying the foundation for H 2 Se donor development, Pluth and co-workers were curious whether this framework could be reengineered to release COSe, providing a new means for the controlled delivery of selenide [84].To test their hypothesis, the authors treated p-fluorophenyl isoselenocyanate with 2-nitrobenzyl alcohol in the presence of NaH to generate lightactivated PhotoSeCM (Figure 7B).To analyze product formation upon photoactivation, the authors used 19 F NMR to streamline the process.Using this method, consumption of PhotoSeCM (−117 ppm) was confirmed but the expected p-fluoroaniline product (due to COSe release) at −130 ppm was not observed.Instead, a signal matching p-fluoroisocyanate was detected, suggesting the direct liberation of selenide rather than COSe serving as an intermediary (Figure 7B).
Interested in assessing a second COSe/H 2 Se-releasing system but without the requirement of photoactivation to simplify the overall mechanism, Pluth and team also examined γ-ketoselenocarbamates, which were reconstituted from their earlier work on γketothiocarbamates [80].This H 2 S donor class undergoes an enol-mediated self-immolating process that delivers COS/H 2 S alongside p-nitroaniline, which can be used to track reaction progress due to its UV-Vis signature at 381 nm.γ-Ketoselenocarbamates were constructed from p-nitrophenyl isoselenocyanate and their activation at different pH values was monitored by UV-Vis.While compound 8, which lacks deprotonatable hydrogens at the β position, failed to generate p-nitroaniline (Figure 7C), compound 9, as expected, showed a pH-dependent rate of release, increasing at higher pH values (Figure 7D).In buffer, the authors were unable to provide direct experimental evidence of selenide release from this donor system as the ensuing p-nitrophenyl isocyanate byproduct is unstable and likely to hydrolyze quickly to p-nitroaniline and CO 2 .Nevertheless, computational studies corroborate the likelihood of direct selenide liberation as it was calculated to be by far the lowest energy decomposition pathway, underscoring the fundamental differences in thiocarbamate/selenocarbamate reactivity.

γ-Ketoselenides
An analogous base-mediated selenide delivery system was first reported by us in 2022 [85].In this study, we prepared a library of γ-ketoselenides (10-13, Figure 8A) upon treatment of the corresponding γ-ketohalide/tosylate with a solution of sodium selenide that was generated in situ by reducing elemental selenium with NaBH 4 .Once in hand, we first monitored the release of selenide from 10 (Figure 8A) in a 1:1 mixture of CD3CN and deuterated phosphate buffer (50 mM, pD 7.4) by tracking the formation of byproduct 10a (Figure 8A) using 1 H NMR. During the experiment, we observed a decrease in the intensity of the protons alpha to selenium while the signal of the terminal enone protons simultaneously increased.The addition of 1,4-dioxane as an internal standard allowed us to track the concentration of both and confirm that the consumption of 10 Once in hand, we first monitored the release of selenide from 10 (Figure 8A) in a 1:1 mixture of CD 3 CN and deuterated phosphate buffer (50 mM, pD 7.4) by tracking the formation of byproduct 10a (Figure 8A) using 1 H NMR. During the experiment, we observed a decrease in the intensity of the protons alpha to selenium while the signal of the terminal enone protons simultaneously increased.The addition of 1,4-dioxane as an internal standard allowed us to track the concentration of both and confirm that the consumption of 10 correlated well with the production of 10a.During these studies, we also observed the formation of a red film in our NMR tube, which we attributed to the rapid oxidation of H 2 Se in an aqueous buffer and its conversion to Se 0 .This was confirmed by 31 P NMR and the formation of triphenylphosphine selenide upon treatment with triphenylphosphine.
Appearing to proceed through an α-deprotonation/β-elimination sequence, we suspected that an enhanced rate of H 2 Se donation from γ-ketoselenides would be observed at both higher pH values and with donors that possess more acidic alpha protons.To test our hypotheses, the same kinetic studies were repeated at pH 6 and 8.5.As anticipated, the rate of selenide donation was found to be intensified under more basic conditions (pH 8.5) and hindered under more acidic conditions (pH 6).Similarly, alpha proton acidity appeared to play a significant role in donor reactivity with 13 undergoing rapid selenide liberation (confirmed with trapping experiments using benzyl bromide), while 11 was found to be considerably more stable than both 10 and 12.In total, these experiments not only verified the release of selenide from γ-ketoselenides but also the mechanism through which it occurs.
The anticancer activity of these compounds was also assessed in HeLa (human cervical cancer) and HCT116 (human colon cancer) cells in culture (Figure 8B).Predictably, 11, which was found to be stable in buffer for months at a time, liberating only trace amounts of selenide, was found to be completely inactive in both cell lines.Conversely, 10, 12, and 13 exhibited low micromolar activity.As a key control, compound 12EC, an oxygen conger of 12, was also tested and found to be completely inactive, confirming that the activity of donors is due to their release of selenide and not the other components of the reaction.It is also worth mentioning that 11, which liberates selenide at an order of magnitude faster than both 10 and 12, was found to be two-fold less potent in both cell lines.This implies that greater antiproliferative activity may be achieved through continuous exposure to low levels of H 2 Se for a prolonged period as opposed to a rapid surge in selenide concentration that is likely afforded by 11.

5 ′ -O-Selenophosphate Nucleosides
It had been previously shown by Kaczmarek and colleagues that 5 ′ -O-thiophosphate nucleosides function as an H 2 S source in the presence of histidine triad nucleotide-binding protein 1 (HINT1) [86,87].Consistent with the common theme throughout this review, Kaczmarek speculated that a selenium congener might function as a HINT1 substrate [88].If so, then perhaps H 2 Se, like H 2 S, would be afforded as a byproduct of the enzyme's hydrolase activity.
To test this premise, 2 ′ -deoxyguanosine-5 ′ -O-selenophosphate (dGMPSe) was produced using a known method for generating phosphoroselenoates [89].After confirming the stability of dGMPSe in the buffer alone, its HINT1-catalyzed hydrolysis was assessed.This selenophosphate derivative was found to be a substrate for HINT1 with both deoxyguanosine monophosphate (dGMP), confirmed by HPLC, and H 2 Se, detected by fluorescence spectroscopy and the use of the reaction-based probe SF7 [90], were observed as products of the enzyme-catalyzed hydrolysis (Figure 9).
After confirming the HINT1-promoted release of H 2 Se in vitro, the cytotoxicity of dGMPSe was evaluated in HeLa cells.Compared to dGMP, which was found to be nontoxic at all concentrations tested, dGMPSe exhibited dose-dependent cytotoxicity with an IC 50 value of 8 µM after a 24 h incubation period.Furthermore, dead cells were shown to exhibit higher fluorescence in the presence of SF7, further supporting the notion that released H 2 Se from dGMPSe is responsible for cell death.duced using a known method for generating phosphoroselenoates [89].After confirming the stability of dGMPSe in the buffer alone, its HINT1-catalyzed hydrolysis was assessed.This selenophosphate derivative was found to be a substrate for HINT1 with both deoxyguanosine monophosphate (dGMP), confirmed by HPLC, and H2Se, detected by fluorescence spectroscopy and the use of the reaction-based probe SF7 [90], were observed as products of the enzyme-catalyzed hydrolysis (Figure 9).After confirming the HINT1-promoted release of H2Se in vitro, the cytotoxicity of dGMPSe was evaluated in HeLa cells.Compared to dGMP, which was found to be nontoxic at all concentrations tested, dGMPSe exhibited dose-dependent cytotoxicity with an IC50 value of 8 µM after a 24 h incubation period.Furthermore, dead cells were shown to exhibit higher fluorescence in the presence of SF7, further supporting the notion that released H2Se from dGMPSe is responsible for cell death.

Nonspecific Electrophilic Traps: Dinitrofluorobenzene, Benzyl Bromide, and Iodoacetamide.
While selective microsensors and a plethora of reaction-based fluorescent probes offer reliable methods for sulfide detection, analogous chemical tools with selenide specificity are lacking.Furthermore, the methylene blue assay, a ubiquitous method for sulfide quantification in buffer, is unlikely to translate to accurate selenide detection due to its rapid oxidation and overall chemical instability compared to that of sulfide.As such, many researchers have turned to the use of nonspecific electrophilic traps as a quick and dirty method for selenide sensing and validation of H2Se donor activation.
As mentioned earlier, Pluth and co-workers relied on dinitrofluorobenzene (DNFB) for confirmation of H2Se donation from TDN1042 (Figure 10A) [65].However, the high electrophilicity of DNFB, coupled with the augmented nucleophilic character of selenium, made this a challenging endeavor.The authors had previously observed the P=Se moiety of the donor reacting directly with other electrophiles, such as benzyl bromide, making it impossible to distinguish donor alkylation from the alkylation of H2Se.Thus, to unequivocally establish selenide release from TDN1042, the authors placed the DNFB trapping solution in a vial separate from the donor.Under this setup, when TDN1042 was acidified with HCl and sparged with N2, the liberated H2Se was volatilized into the headspace and bubbled through the separate trapping solution containing an excess of DNFB.The contents of the trapping solution were then analyzed by HPLC with the expected mono and diselenide products being clearly visible (Figure 10A).While selective microsensors and a plethora of reaction-based fluorescent probes offer reliable methods for sulfide detection, analogous chemical tools with selenide specificity are lacking.Furthermore, the methylene blue assay, a ubiquitous method for sulfide quantification in buffer, is unlikely to translate to accurate selenide detection due to its rapid oxidation and overall chemical instability compared to that of sulfide.As such, many researchers have turned to the use of nonspecific electrophilic traps as a quick and dirty method for selenide sensing and validation of H 2 Se donor activation.
As mentioned earlier, Pluth and co-workers relied on dinitrofluorobenzene (DNFB) for confirmation of H 2 Se donation from TDN1042 (Figure 10A) [65].However, the high electrophilicity of DNFB, coupled with the augmented nucleophilic character of selenium, made this a challenging endeavor.The authors had previously observed the P=Se moiety of the donor reacting directly with other electrophiles, such as benzyl bromide, making it impossible to distinguish donor alkylation from the alkylation of H 2 Se.Thus, to unequivocally establish selenide release from TDN1042, the authors placed the DNFB trapping solution in a vial separate from the donor.Under this setup, when TDN1042 was acidified with HCl and sparged with N 2 , the liberated H 2 Se was volatilized into the headspace and bubbled through the separate trapping solution containing an excess of DNFB.The contents of the trapping solution were then analyzed by HPLC with the expected mono and diselenide products being clearly visible (Figure 10A).Likewise, Yi and colleagues first utilized iodoacetamide for quick confirmation of selenide donation from their selenocyclopropenone-based donors (Figure 10B) [68].This analysis was further complicated by the fact that cysteine, which can be consumed by the added iodoacetamide, was used to trigger H2Se release from this donor class.Nevertheless, using HRMS the authors observed the corresponding mono and diselenide products, Likewise, Yi and colleagues first utilized iodoacetamide for quick confirmation of selenide donation from their selenocyclopropenone-based donors (Figure 10B) [68].This analysis was further complicated by the fact that cysteine, which can be consumed by the added iodoacetamide, was used to trigger H 2 Se release from this donor class.Nevertheless, using HRMS the authors observed the corresponding mono and diselenide products, which is consistent with the release of selenide and the autooxidation process.
We, too, relied on nonspecific electrophiles, both benzyl bromide and iodoacetamide, for confirmation of H 2 Se liberation from our γ-ketoselenide-based system [85].Like the Pluth group, we also sought to unambiguously confirm H 2 Se donation from 10 by trapping the gas in a separate vial (Figure 10C).We elected to use iodoacetamide for these experiments as the ensuing product from its trapping of volatilized H 2 Se would be easily recognizable by HRMS.Indeed, when 10 was acidified and sparged with argon, released H 2 Se was transferred through a cannula needle and into a separate trapping solution where the expected selenide product was clearly visible by HRMS.

Fluorescent Sensors Based on Benzoselenadiazole Se-N Bond Cleavage
A small molecule fluorescent probe with high selectivity towards H 2 Se was first reported by Tang and colleagues in 2016 (NIR-H 2 Se, Figure 11A) [91].By fabricating a benzoselenadiazole moiety onto a mercaptan dye, the authors found that Se-N bond cleavage occurred quickly in the presence of H 2 Se, but not when exposed to other selenols or thiols, to afford to a fluorescent diamine reporter (λ ex : 688 nm, λ em : 735 nm).With an H2Se-selective sensor in hand, the authors then used NIR-H2Se to monitor cellular H2Se levels in HepG2 cells using Na2SeO3 as a metabolic precursor.In this experiment, a dose and time-dependent increase in fluorescence was observed, as expected.Moreover, these investigations were conducted under both hypoxic (1% pO2) and normoxic (20% O2) conditions, with a significant reduction in fluorescence being observed during the la er.This was a ributed to rapid H2Se oxidation and the production of superoxide and other reactive oxygen species while exposed to higher O2 levels.Based on these observations, it was concluded that the anticancer activity of Na2SeO3 in HepG2 cells under normoxic conditions can be a ributed to ROS-induced cell death.Conversely, under hypoxic conditions, which is a hallmark of solid tumors, a non-oxidative stress mechanism is likely in play due to a notable buildup in cellular H2Se.This hypothesis was further validated in a solid tumor mouse model using NIR-H2Se.
A lysosomal-specific H2Se sensor based on this same framework was reported by Zhang and Jing in 2019 (Se-1, Figure 11B) [92].In addition to a benzoselenadiazole moiety for selective H2Se detection, a morpholino group was appended to the sensor for lysosomal-targeting [93].Under simulated lysosomal conditions (acetate buffer, pH 5), a notable increase in fluorescence at 535 nm (λex: 435 nm) was observed when a solution of Se-1 was exposed to H2Se (but not other biologically relevant analytes).Moreover, the fluorescent With an H 2 Se-selective sensor in hand, the authors then used NIR-H 2 Se to monitor cellular H 2 Se levels in HepG2 cells using Na 2 SeO 3 as a metabolic precursor.In this experiment, a dose and time-dependent increase in fluorescence was observed, as expected.Moreover, these investigations were conducted under both hypoxic (1% pO 2 ) and normoxic (20% O 2 ) conditions, with a significant reduction in fluorescence being observed during the latter.This was attributed to rapid H 2 Se oxidation and the production of superoxide and other reactive oxygen species while exposed to higher O 2 levels.Based on these observations, it was concluded that the anticancer activity of Na 2 SeO 3 in HepG2 cells under normoxic conditions can be attributed to ROS-induced cell death.Conversely, under hypoxic conditions, which is a hallmark of solid tumors, a non-oxidative stress mechanism is likely in play due to a notable buildup in cellular H 2 Se.This hypothesis was further validated in a solid tumor mouse model using NIR-H 2 Se.
A lysosomal-specific H 2 Se sensor based on this same framework was reported by Zhang and Jing in 2019 (Se-1, Figure 11B) [92].In addition to a benzoselenadiazole moiety for selective H 2 Se detection, a morpholino group was appended to the sensor for lysosomaltargeting [93].Under simulated lysosomal conditions (acetate buffer, pH 5), a notable increase in fluorescence at 535 nm (λ ex : 435 nm) was observed when a solution of Se-1 was exposed to H 2 Se (but not other biologically relevant analytes).Moreover, the fluorescent enhancement of Se-1 in the presence of H 2 Se was found to be consistent within a pH range of 4.5-7, confirming its compatibility with lysosomal conditions.
After establishing its reactivity and photophysical properties in buffer, lysosomaltargeting of Se-1 was confirmed in HepG2 cells using LysoTracker Blue (Pearson's correlation coefficient of 0.91).Additionally, using Se-1, the authors observed elevated levels of lysosomal H 2 Se in hypoxic HepG2 cells, whereas little lysosomal fluorescence was observed under normoxic conditions.
To date, benzoselenadiazoles have not been used as a recognition subunit to validate selenide delivery from novel donor scaffolds.However, its reported high reactivity towards H 2 Se could prove useful in future studies as the transient nature of H 2 Se requires rapid detection for accurate monitoring.

Fluorescent Sensors Based on Disulfide Bond Cleavage
A second-generation sensor from Tang and co-workers employed disulfide bond reduction as a recognition mechanism for H 2 Se-initiated turn-on fluorescence (Hcy-H 2 Se, Figure 12) [94].Thiol-activated prodrugs and chemosensors that utilize disulfide bond reduction as their initiation step have been widely reported [95].Based on these accounts, it was suspected that a more stable cyclic disulfide (i.e., a six-membered ring) would respond much more quickly to H 2 Se, given its heightened nucleophilicity, thereby imparting selectivity over other selenols and thiols.
Molecules 2024, 29, x FOR PEER REVIEW 15 of 21 reduction as their initiation step have been widely reported [95].Based on these accounts, it was suspected that a more stable cyclic disulfide (i.e., a six-membered ring) would respond much more quickly to H2Se, given its heightened nucleophilicity, thereby imparting selectivity over other selenols and thiols.Hcy-H2Se was generated by combining oxidized dithiothreitol [96] with a masked hemicyanine dye via a carbamate linker.When exposed to H2Se, a maximum fluorescence intensity (λex: 470 nm, λem: 535 nm) was reached almost immediately, indicating rapid H2Se-initiated disulfide bond reduction and cyclization (Figure 12).Selectivity studies indicated that the addition of glutathione, H2S, selenocysteine, dithiothreitol, bovine serum albumin, and thioredoxin reductase yielded li le fluorescence compared to H2Se.Moreover, the emission intensity at 535 nm correlated well with increasing concentrations of H2Se, affording a good linear relationship between the two.
Bioimaging of H2Se in live human cells was accomplished with Hcy-H2Se [94].HepG2 cells exposed to Na2SeO3 and Hcy-H2Se under hypoxic conditions exhibited fluorescence that was both dose-and time-dependent.The authors also demonstrated that hypoxic tumor regions in mice injected with sodium selenite could be imaged with Hcy-H2Se.This further supports the anticancer effects of selenium in hypoxic solid tumors being due to an increase in reductive rather an oxidative stress.

Spectroscopic Sensors Based on Nucleophilic Substitution
While examining selenocyclopropenones and selenoamides as potential cysteine-activated donors (Figure 6), Yi and collaborators developed a quantitative assay for H2Se Hcy-H 2 Se was generated by combining oxidized dithiothreitol [96] with a masked hemicyanine dye via a carbamate linker.When exposed to H 2 Se, a maximum fluorescence intensity (λ ex : 470 nm, λ em : 535 nm) was reached almost immediately, indicating rapid H 2 Se-initiated disulfide bond reduction and cyclization (Figure 12).Selectivity studies indicated that the addition of glutathione, H 2 S, selenocysteine, dithiothreitol, bovine serum albumin, and thioredoxin reductase yielded little fluorescence compared to H 2 Se.Moreover, the emission intensity at 535 nm correlated well with increasing concentrations of H 2 Se, affording a good linear relationship between the two.
Bioimaging of H 2 Se in live human cells was accomplished with Hcy-H 2 Se [94].HepG2 cells exposed to Na 2 SeO 3 and Hcy-H 2 Se under hypoxic conditions exhibited fluorescence that was both dose-and time-dependent.The authors also demonstrated that hypoxic tumor regions in mice injected with sodium selenite could be imaged with Hcy-H 2 Se.This further supports the anticancer effects of selenium in hypoxic solid tumors being due to an increase in reductive rather an oxidative stress.

Spectroscopic Sensors Based on Nucleophilic Substitution
While examining selenocyclopropenones and selenoamides as potential cysteineactivated donors (Figure 6), Yi and collaborators developed a quantitative assay for H 2 Se based on its nucleophilic substitution with a carefully chosen electrophilic species [68].Upon testing several potential candidates, the authors discovered that commercially available Cy7-Cl reacted much more quickly with selenide than other selenols and thiols, to form Cy7-SeH (Figure 13A).As the reaction progressed, it was noted that the starting absorbance of 780 nm (Cy7-Cl) was shifted to 710 nm (Cy7-SeH), offering a convenient colorimetric method for monitoring reaction progress.The reaction between Cy7-Cl (500 µM) and various amounts of Na 2 Se (25-350 µM) was also monitored HPLC, with a plot of Cy7-SeH peak area vs. selenide concentration yielding a straight line.This calibration curve was then used to effectively determine the H 2 Se-releasing efficiency from selenocyclopropenones in the presence of cysteine.While evaluating the H2Se-releasing efficiencies of cyclic-PSe donors (Figure 5), the Pluth group found that commercially available 4-chloro-7-nitrobenzofurazan (NBD-Cl), which had been previously used to detect H2S [97], could also be used to trap selenide via a nucleophilic aromatic substitution reaction (Figure 13B) [66].Initially, the authors analyzed the reaction between NBD-Cl and tetrabutylammonium hydroselenide (NBu4SeH) in PBS (pH 7.4).They found that substoichiometric amounts of selenide provided NBD2Se (λabs: 428 nm), while stoichiometric HSe -yielded NBD-SeH as the primary product (λabs: 551 nm).With confirmation of a colorimetric response, NBD-Cl was then used to monitor the hydrolysis of Cat-PSe in PBS (Figure 13C).While the formation of NBD-SeH was clearly visible (λabs: 551 nm), the rate of apparent H2Se liberation appeared to occur much faster when compared to earlier 31 P NMR hydrolysis experiments.The authors a ributed this notable rate enhancement to donor alkylation, rather than hydrolysis, while in the While evaluating the H 2 Se-releasing efficiencies of cyclic-PSe donors (Figure 5), the Pluth group found that commercially available 4-chloro-7-nitrobenzofurazan (NBD-Cl), which had been previously used to detect H 2 S [97], could also be used to trap selenide via a nucleophilic aromatic substitution reaction (Figure 13B) [66].Initially, the authors analyzed the reaction between NBD-Cl and tetrabutylammonium hydroselenide (NBu 4 SeH) in PBS (pH 7.4).They found that substoichiometric amounts of selenide provided NBD 2 Se (λ abs : 428 nm), while stoichiometric HSe -yielded NBD-SeH as the primary product (λ abs : 551 nm).With confirmation of a colorimetric response, NBD-Cl was then used to monitor the hydrolysis of Cat-PSe in PBS (Figure 13C).While the formation of NBD-SeH was clearly visible (λ abs : 551 nm), the rate of apparent H 2 Se liberation appeared to occur much faster when compared to earlier 31 P NMR hydrolysis experiments.The authors attributed this notable rate enhancement to donor alkylation, rather than hydrolysis, while in the presence of a strong electrophile, such as NBD-Cl.Therefore, while the use of NBD-Cl appears to provide a convenient colorimetric method for monitoring donor progress, additional trapping experiments are likely necessary to untangle donor alkylation from the alkylation of released H 2 Se in solution.

Fluorescent Sensors Based on Azide Reduction
Like H 2 S-responsive NBD-Cl being reintroduced for H 2 Se detection, H 2 S-sensitive fluorescent sensors that rely on aryl azide reduction were reexamined for their capacity to detect H 2 Se [89].Probes SF4 and SF7 (Figure 14) were previously engineered by Chang and co-workers for monitoring H 2 S levels under physiological conditions [90,98].While stable towards other sulfur-containing molecules, H 2 S initiates aryl azide reduction on the masked rhodamine dye, resulting in a turn-fluorescent response (Figure 14A).Given the increased reactivity of H2Se, Kaczmarek and co-workers suspected that these same sensors would be responsive towards H2Se and used them to confirm selenide release from dGMPSe in both in a buffer and in live HeLa cells (Figure 14B) [88].In fact, when using SF7 and SF4, higher fluorescence values were observed with dGMPSe compared to its sulfur congener, which likely stems from the higher reactivity and reductive properties of H2Se compared to that of H2S.Thus, while not selective for H2Se, aryl azide reduction appears to be a potential method for the real-time monitoring of donor progress.

Conclusions and Outlook
The continued evolution of H2Se-specific chemical tools is a prime objective for those interested in uncovering the (patho)physiological effects of hydrogen selenide.While donor compounds capable of providing the slow and sustained release of H2Se have begun to emerge, the introduction of additional stimulus-responsive donors (i.e., enzyme or bioanalyte-triggered) with greater spatiotemporal control over their biological delivery of H2Se will serve as a great advancement in the field.H2Se detection and quantification, especially when liberated from a donor scaffold, remains a great challenge.Most current methods rely on electrophiles to trap the released selenide, which often leads to interference from the donor itself given the intensified nucleophilic character of selenium.New approaches for detection that avoid the introduction of strong electrophiles should continue to be explored, including additional reaction-based fluorescent sensors that can be used in cellular and in vivo imaging experiments.Together, these compounds will continue to unearth the unique aspects of H2Se chemical biology, while providing new evi- Given the increased reactivity of H 2 Se, Kaczmarek and co-workers suspected that these same sensors would be responsive towards H 2 Se and used them to confirm selenide release from dGMPSe in both in a buffer and in live HeLa cells (Figure 14B) [88].In fact, when using SF7 and SF4, higher fluorescence values were observed with dGMPSe compared to its sulfur congener, which likely stems from the higher reactivity and reductive properties of H 2 Se compared to that of H 2 S. Thus, while not selective for H 2 Se, aryl azide reduction appears to be a potential method for the real-time monitoring of donor progress.

Conclusions and Outlook
The continued evolution of H 2 Se-specific chemical tools is a prime objective for those interested in uncovering the (patho)physiological effects of hydrogen selenide.While donor compounds capable of providing the slow and sustained release of H 2 Se have begun to emerge, the introduction of additional stimulus-responsive donors (i.e., enzyme or bioanalyte-triggered) with greater spatiotemporal control over their biological delivery of H 2 Se will serve as a great advancement in the field.H 2 Se detection and quantification, especially when liberated from a donor scaffold, remains a great challenge.Most current methods rely on electrophiles to trap the released selenide, which often leads to interfer-ence from the donor itself given the intensified nucleophilic character of selenium.New approaches for detection that avoid the introduction of strong electrophiles should continue to be explored, including additional reaction-based fluorescent sensors that can be used in cellular and in vivo imaging experiments.Together, these compounds will continue to unearth the unique aspects of H 2 Se chemical biology, while providing new evidence that further supports (or refutes) the addition of H 2 Se as the fourth gasotransmitter.

Figure 4 .
Figure 4. Proposed hydrolytic pathway of TDN1042, which results in the release of two equivalents of H2Se and the formation of phenylphosphonic acid.

Figure 4 .
Figure 4. Proposed hydrolytic pathway of TDN1042, which results in the release of two equivalents of H 2 Se and the formation of phenylphosphonic acid.

Figure 5 .
Figure 5. (A) Cyclic-PSe donors arranged in order of decreasing rates of hydrolysis; (B) Proposed hydrolytic pathway of cyclic-PSe donors, which results in the release of one equivalent of H2Se and the formation of phenylphosphonic acid.

Figure 5 .
Figure 5. (A) Cyclic-PSe donors arranged in order of decreasing rates of hydrolysis; (B) Proposed hydrolytic pathway of cyclic-PSe donors, which results in the release of one equivalent of H 2 Se and the formation of phenylphosphonic acid.

Figure 7 .
Figure 7. (A) General strategy for generating stimuli-responsive COS/H 2 S donors.(B) Proposed mechanism for the direct release of H 2 Se from PhotoSeCM upon irradiation at 365 nm.(C) Control compound used to highlight the stability of γ-ketoselenocarbamates in water.(D) Proposed mechanism for the direct release of H 2 Se from γ-ketoselenocarbamates containing a deprotonatable hydrogen at the β position.

Molecules 2024 , 21 Figure 8 .
Figure 8. (A) A library of γ-ketoselenides that undergo base-promoted ⍺-deprotonation/β-elimination to release H2Se.(B) Cell growth inhibition of HeLa and HCT116 cells in culture.IC50 values were determined after a 24 h incubation period with donor.

Figure 8 .
Figure 8. (A) A library of γ-ketoselenides that undergo base-promoted α-deprotonation/βelimination to release H 2 Se.(B) Cell growth inhibition of HeLa and HCT116 cells in culture.IC 50 values were determined after a 24 h incubation period with donor.

Figure 10 .
Figure 10.Use of nonspecific electrophilic traps to confirm H 2 Se release from donors ((A) TDN1042, (B) 1, (C) 10) by forming stable selenide/diselenide products that can be easily identified by spectroscopic methods.

Figure 11 .
Figure 11.(A) Reaction-based fluorescent sensor with selectivity towards H 2 Se based on benzoselenadiazole Se-N bond cleavage.(B) A lysosomal-targeting fluorescent sensor for H 2 Se bioimaging.

Figure 12 .
Figure 12.Proposed mechanism for Hcy-H 2 Se turn-on fluorescence initiated by H 2 Se-promoted disulfide reduction.

Molecules 2024 , 21 Figure 13 .
Figure 13.(A,B) Commercially available electrophilic traps that provide a colorimetric readout for monitoring H2Se donor progress.(C) Cautioning researchers that complementary H2Se measurements should be employed to avoid confusing donor alkylation with the alkylation of released H2Se in solution.

Figure 13 .
Figure 13.(A,B) Commercially available electrophilic traps that provide a colorimetric readout for monitoring H 2 Se donor progress.(C) Cautioning researchers that complementary H 2 Se measurements should be employed to avoid confusing donor alkylation with the alkylation of released H 2 Se in solution.

Molecules 2024 ,
29, x FOR PEER REVIEW 17 of 21stable towards other sulfur-containing molecules, H2S initiates aryl azide reduction on the masked rhodamine dye, resulting in a turn-fluorescent response (Figure14A).

Figure 14 .
Figure 14.(A) Aryl azide reduction for H2S-initiated turn-on fluorescence.(B) The use of aryl azides as fluorescent probes for tracking H2Se liberation from donor compounds.

Figure 14 .
Figure 14.(A) Aryl azide reduction for H 2 S-initiated turn-on fluorescence.(B) The use of aryl azides as fluorescent probes for tracking H 2 Se liberation from donor compounds.